Note: Descriptions are shown in the official language in which they were submitted.
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CA 02604761 2007-10-12
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TITLE OF THE INVENTION
REAL-TIME HPV PCR ASSAYS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/675,938 filed
Apri128, 2005, the contents of which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
The present invention relates generally to PCR-based assays to detect the
presence of
human papilloinavirus (HPV) types in clinical samples. More specifically, it
relates to fluorescent
multiplex PCR assays, wherein multiple fluorophores are used to simultaneously
detect a plurality of
HPV loci in a single PCR reaction tube.
BACKGROUND OF THE INVENTION
More than 80 types of human papillomaviruses (HPVs) have been identified. The
different types of HPV cause a wide variety of biological phenotypes, from
benign proliferative warts to
malignant carcinomas (for review, see McMurray et al., Int. J. Exp. Pathol.
82(1): 15-33 (2001)). HPV6
and HPV 11 are the types most commonly associated with benign warts, whereas
HPV 16 and HPV 18 are
the high-risk types most frequently associated with malignant lesions.
Determination of the specific type
of HPV in a clinical sample is, therefore, critical for predicting risk of
developing HPV-associated
disease.
Several nucleic acid-based methods have been utilized to identify and quantify
specific
HPV types in clinical samples, such as detection of viral nucleic acid by in
situ hybridization, Southern
blot analysis, hybrid capture or polymerase chain reaction (PCR). The Hybrid
Capture II (Digene
Diagnostics, Inc., Gaithersburg, MD) assay utilize antibody capture and non-
radioactive signal detection,
but detect only a single target of a given set of HPV types (See, e.g., Clavel
et al., British J. Cafacer
80(9): 1306-11 (1999)). Additionally, because The Hybrid Capture II assay
uses a cocktail of RNA
probes (probe cocktails are available for high risk or low-risk HPV types), it
does not provide
information as to the specific HPV type detected in a sample, but rather
provides only a positive or
negative for the presence of high-risk or low-risk HPV. Similarly, many PCR-
based metliods often
involve amplification of a single specific HPV target sequence followed by
blotting the resulting
amplicon to a membrane and probing with a radioactively labeled
oligonucleotide probe.
Other methods exploit the high homology between specific HPV genes of
different types
through the use of commercially available consensus primers capable of PCR
amplifying numerous HPV
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types present in a sample. The presence of a specific HPV type is then
identified using a type-specific
oligonucleotide probe. See, e.g., Kleter et al., Journal of Clinical
Microbiology 37(8): 2508-2517
(1999); Gravitt et al., Journal of Clinical Microbiology 38(1): 357-361
(2000). Similarly, assays that
utilize degenerate PCR primers take advantage of the homology between HPV
types, allowing detection
of a greater number of HPV types than methods utilizing specific primer sets.
See, e.g. Harwood et al.,
Journal of Clinical Mici=obiology 37(11): 3545-3555 (1999). Such assays also
require additional
experimentation to identify specific HPV types.
The PCR methods described above can be associated with several problems. For
exarimple, differences in reaction efficiencies among HPV types can result in
disproportionate
amplification of some types relative to others. Additionally, the equilibrium
for amplification will be
driven towards those types that exist at higher copy numbers in a sample,
which will consume the PCR
reaction components, thus making amplification of the minor HPV types less
likely.
Also described in the art is a 5' exonuclease fluorogenic PCR-based assay (Taq-
Man
PCR) which allows detection of PCR products in real-time and eliminates the
need for radioactivity. See,
e.g., U.S. Patent No. 5,538,848; Holland et al, Proe. Natl. Acaa'. Sci. USA
88: 7276-7280 (1991). This
method utilizes a labeled probe, comprising a fluorescent reporter
(fluorophore) and a quencher that
hybridizes to the target DNA between the PCR primers. Excitation of the
fluorophore results in the
release of a fluorescent signal by the fluorophore which is quenched by the
quencher. Amplicons can be
detected by the 5' - 3' exonuclease activity of the TAQ DNA polymerase, which
degrades double-
stranded DNA encountered during extension of the PCR primer, thus releasing
the fluorophore from the
probe. Thereafter, the fluorescent signal is no longer quenched and
accumulation of the fluorescent
signal, which is directly correlated with the amount of target DNA, can be
detected in real-time with an
automated fluorometer.
Taq-Man PCR assays have been adapted for HPV type detection. Swan et al.
(Journal of
Clinical Microbiology 35(4): 886-891 (1997)) disclose a fluorogenic probe
assay that utilizes type-
specific HPV primers that amplify a portion of the Ll gene in conjunction with
type-specific probes.
The Swan et al. assay measures fluorescent signal at the end of a fixed number
of PCR cycles (endpoint
reading) and not in real-time.
Josefsson et al. (Journal of Clinical Microbiology 37(3): 490-96 (1999))
report a Taq-
Man assay that targets a higlily conserved portion of the El gene in
conjunction with type-specific probes
labeled with different fluorescent dyes. A number of HPV types were amplified
by utilizing a mixture of
specific and degenerate primers. Josefsson et al. utilized up to three type-
specific probes per assay,
which were designed to detect a portion of the El gene from different HPV
types. Unlike the Swan et al.
assay, Josefsson et al. measured the accumulation of fluorescence in real-
time.
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Tucker et al. (Molecular Diagnosis 6(1): 39-47 (2001)) describe an assay that
targets a
conserved region spanning the E6/E7 junction. Like the Josefsson assay, Tucker
et al. employed real-
time detection and type-specific fluorescent probes. Tucker et al. also
utilized multiplex PCR to
simultaneously detect HPV target sequences and either the actin or globin
cellular loci in the same
reaction tube.
The methods described above typically involve testing for the presence of a
single viral
locus in a DNA sample such as the L1 locus. A disadvantage of single-locus
assays is that the high
degree of homology among specific HPV genes from one HPV type to another leads
to an excessive
occurrence of false positive results. This level of homology makes it
difficult to design a PCR assay that
is specific for a single HPV type. It is therefore necessary to confirm
positive results by testing for the
presence of several loci of a single HPV-type. The further experimentation
required to verify positive
results is cumbersome and time-consuming. Establishment of the HPV status of a
clinical sample for
four different HPV types typically consumes 26-30 man-hours.
Single-locus assays may also lead to false negative results. It is well
established that the
relationship between the HPV genome and chromosomal host DNA may change during
the multistage
tumorigenic process (For review, see McMurray et al., Int. J. Exp. Path. 82:
15-33 (2001)). Premalignant
lesions are often associated with episomal forms of HPV DNA while later-stage
tumors typically have
integrated HPV sequences. As a result of the integration correlated with
advanced stages of disease
progression, the open reading frame of specific HPV genes, such as the Ll
gene, may become disrupted.
Such disruption of HPV gene sequences may lead to false negative results in
assays that target the
disrupted sequence.
Multiplex assays describing the siinultaneous identification of a plurality of
HPV genes
from a single HPV type are described in WO 03/019143. However, these assays
are specifically directed
to the identification of HPV types 6, 11, 16, and 18.
Despite the development of the HPV assays described above, it would be
advantageous
to develop an assay that is highly sensitive and reproducible, and that
requires reduced man-hours
compared to methods disclosed in the art. It would also be advantageous to
develop an assay for the
identification of additional HPV types, specifically HPV types that are
associated with a pathological
phenotype.
SUMMARY OF THE INVENTION
The present invention relates to a fluorescent multiplex PCR assay for
detecting the
presence of an HPV type in a sample which uses multiple fluorophores to
simultaneously detect a
plurality of HPV loci of the same HPV type, wherein the HPV type is selected
from the group consisting
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of HPV33, HPV35, HI.'V39, HPV51, HPV56, and HPV59. Said BPV types have been
associated with
an oncogenic phenotype.
More specifically, the present invention relates to a method for detecting the
presence of
a nucleic acid of a hunian papillomavirus (HPV) type in a nucleic acid-
containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and a
plurality
of oligonucleotide sets to produce a plurality of PCR amplicons;
wherein each oligonucleotide set consists of (a) a forward discriminatory PCR
primer
hybridizing to a first location of a nucleic acid sequence of an HPV type, (b)
a reverse discriminatory
PCR primer hybridizing to a second location of the nucleic acid sequence of
the HPV type downstream
of the first location, and (c) a fluorescent probe labeled with a quencher
molecule and a fluorophore
which emits energy at a unique emission maxima; said probe hybridizing to a
location of the nucleic acid
sequence of the BPV type between the first and the second locations;
wherein each oligonucleotide set specifically hybridizes to a different HPV
amplicon
derived from the same HPV type, and wlierein the HPV type is selected from the
group consisting of:
HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59.
allowing said nucleic acid polymerase to digest each fluorescent probe during
amplification to dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV type if a change of
fluorescence is
detected in at least two emission maxima.
In a preferred embodiment of this invention, each oligonucleotide set of the
plurality of
oligonucleotide sets is specific to a single gene of the HPV type to be
detected. In other words, each
oligonucleotide set of the method of the present invention hybridizes to
nucleotide sequences derived
from a single BPV gene of the same type. For example, the oligonucleotide
priuuers and probe of a first
oligonucleotide set hybridize to the E6 gene, the oligonucleotide primers and
probe of a second
oligonucleotide set hybridize to the E7 gene and the oligonucleotide primers
and probe of a third
oligonucleotide set hybridize to the L1 gene. As a result, a plurality of PCR
amplicons is created
wherein each PCR amplicon is specific to a single BPV gene of the HPV type to
be detected.
In an alternative embodiment of this invention, the forward discriminatory PCR
primer
and the reverse discriminatory PCR primer of at least one oligonucleotide set
are specific to a different
gene of the same BPV type. For example, a forward discriminatory primer
hybridizes to the E6 gene and
a reverse discriminatory primer hybridizes to the E7 gene. As a result, at
least one PCR amplicon
comprises a sequence of nucleotides derived from more than one gene. The
oligonucleotide probe
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specific to said amplicon may hybridize, for example, to a sequence of
nucleotides derived from the E6
gene, a sequence of nucleotides derived from the E7 gene, or a sequence of
nucleotides that crosses the
E6/E7 boundary.
In a preferred embodiment of this invention, the HPV type is selected from the
group
consisting of: HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59.
In a further preferred embodiment of the method of the present invention, the
number of
oligonucleotide sets is two and the oligonucleotide sets specifically
hybridize to the E6 and E7 genes of
HPV. A sample is positive for the HPV type being tested if both of the E6 and
E7 genes are detected.
Another embodiment of this invention relates to an oligonucleotide probe
comprising a
sequence of nucleotides specific to a single HPV type. Said oligonucleotide
probe can bind to specific
HPV amplicons resulting from PCR amplification of viral DNA using specific
oligonucleotide primers.
In a further embodiment of this invention, said oligonucleotide probe
comprises a sequence of
nucleotides selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID
NO:34, SEQ ID NO: 35, and SEQ ID NO:36.
The present invention also relates to said oligonucleotide probes further
comprising a
fluorophore and a quencher molecule. In a preferred embodiment of this
invention, the fluorophore is
selected from the group consisting of: FAMTM, JOETM, TETTM, (Applera Corp.,
Norwalk, CT) and CAL
Flour Orange (Biosearch Technologies Inc., Novato, CA) and the quencher is
non-fluorescent. In an
especially preferred embodiment of this invention, the quencher is BHQTMI
(Biosearch Technologies).
The present invention further relates to a primer pair for the PCR
amplification of HPV
nucleic acid, wherein both the forward and reverse PCR primers are
discriminatory (see FIGURE 1). In a
preferred embodiment of the invention, the nucleotide sequences of the primer
pair are selected from the
group consisting of: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4,
SEQ ID NO:5
and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10,
SEQ ID NO: 11
and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID
NO:16, SEQ ID
NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID
NO:22, and
SEQ ID NO:23 and SEQ ID NO:24.
As used herein, the term "oligonucleotide" refers to linear oligomers of
natural or
modified monomers or linkages, including deoxyribonucleosides,
ribonucleosides, and the like, capable
of specifically binding to a target polynucleotide by way of a regular pattern
of monomer-to-monomer
interactions, such as Watson-Crick type base pairing. For purposes of this
invention, the term
oligonucleotide includes both oligonucleotide probes and oligonucleotide
primers.
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As used herein, the term "primer" refers to an oligonucleotide that is capable
of acting as
a point of initiation of synthesis along a complementary strand when placed
under conditions in which
synthesis of a primer extension product which is complementary to a nucleic
acid strand is catalyzed.
Such conditions include the presence of four different deoxyribonucleoside
triphosphates and a
polymerization-inducing agent such as DNA polymerase or reverse transcriptase,
in a suitable buffer
("buffer" includes components which are cofactors, or which affect ionic
strength, pH, etc.), and at a
suitable temperature. As employed herein, an oligonucleotide primer can be
naturally occurring, as in a
purified restriction digest, or be produced synthetically. The primer is
preferably single-stranded for
maximum efficiency in amplification.
As used herein, "primer pair" refers to two primers, a forward primer and a
reverse
primer, that are capable of participating in PCR amplification of a segment of
nucleic acid in the
presence of a nucleic acid polymerase to produce a PCR amplicon. The primers
that comprise a primer
pair can be specific to the same HPV gene, resulting in an amplicon that
consists of a sequence of
nucleotides derived from a single HPV gene. Alternatively, the primers that
comprise a primer pair can
be specific to different HPV genes that reside within close proximity to each
other within the HPV
genome, thereby producing amplicons that consist of a sequence of nucleotides
derived from more than
one gene.
As used herein, "unique," in reference to the fluorophores of the present
invention,
means that each fluorophore emits energy at a differing emission maxima
relative to all other
fluorophores used in the particular assay. The use of fluorophores with unique
emission maxima allows
the simultaneous detection of the fluorescent energy emitted by each of the
plurality of fluorophores used
in the particular assay.
As used herein, the term "discriminatory," used in reference to the
oligonucleotide
primers and probes of the present invention, means that said primers and
probes are specific to a single
HPV type. It includes HPV primers and probes specific to a single HPV type,
but that share some
homology with other HPV types. "Discriminatory" primers and probes of the
present invention include
those oligonucleotides that lack 3' homology with other HPV types in at least
one nucleotide or more.
Such a residue that is unique for the specific HPV type at the specific
position and acts to discriminate
the HPV type from the others in the alignment referred to as a "discriminatory
base". The term
"discriminatory," in reference to oligonucleotides, does not include primers
and probes that are specific
to more than one HPV type, i.e. those that share full homology with greater
than one HPV type.
As used herein, "amplicon" refers to a specific product of a PCR reaction,
which is
produced by PCR amplification of a sample comprising nucleic acid in the
presence of a nucleic acid
polymerase and a specific primer pair. An amplicon can consist of a nucleotide
sequence derived from a
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single gene of a single HPV type or an amplicon can consist of a nucleotide
sequence derived from more
than one gene of a single HPV type.
As used herein, "oligonucleotide set" refers to a grouping of a pair of
oligonucleotide
primers and an oligonucleotide probe that hybridize to a specific nucleotide
sequence of a single HPV
type. Said oligonucleotide set consists of: (a) a forward discriminatory
primer that hybridizes to a first
location of a nucleic acid sequence of an HPV type; (b) a reverse
discrim.inatory primer that hybridizes to
a second location of the nucleic acid sequence of the HPV type downstream of
the first location and (c) a
fluorescent probe labeled with a fluorophore and a quencher, which hybridizes
to a location of the
nucleic acid sequence of the HPV type between the primers. In other words, an
oligonucleotide set
consists of a set of specific PCR primers capable of initiating synthesis of
an amplicon specific to a
single HPV type, and a fluorescent probe which hybridizes to the amplicon.
As used herein, "plurality" means two or more.
As used herein, "specifically hybridizes," in reference to oligonucleotide
sets,
oligonucleotide primers, or oligonucleotide probes, means that said
oligonucleotide sets, primers or
probes hybridize to a nucleic acid sequence of a single HPV type.
As used herein, "gene" means a segment of nucleic acid involved in producing a
polypeptide chain. It includes both translated sequences (coding region) and
5' and 3' untranslated
sequences (non-coding regions) as well as intervening sequences (introns)
between individual coding
segments (exons). For purposes of this invention, the HPV genome has nine
genes: Ll, L2, and El - E7.
As used herein, "locus" refers to the position on a chromosome at which the
gene for a
trait resides. The term locus includes any one of the alleles of a specific
gene. It also includes
homologous genes from different HPV types. For example, PCR assays that detect
the L1 gene in
HPV16 and HPV6 are single-locus assays, despite the detection of sequences
from different HPV types.
Contrarily, for example, assays that detect the Ll gene and the El gene of a
single HPV type are multiple
locus assays, even though a single HPV type is detected.
As used herein, "HPV" means human papillomavirus. "IiPV" is a general term
used to
refer to any type of HPV, whether currently known or subsequently described.
As used herein, "fluorophore" refers to a fluorescent reporter molecule which,
upon
excitation with a laser, tungsten, mercury or xenon lamp, or a light emitting
diode, releases energy in the
form of light with a defined spectrum. Through the process of fluorescence
resonance energy transfer
(FRET), the light emitted from the fluorophore can excite a second molecule
whose excitation spectrum
overlaps the emission spectrum of the fluorophore. The transfer of emission
energy of the fluorophore to
another molecule quenches the emission of the fluorophore. The second molecule
is known as a
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quencher molecule. The term "fluorophore" is used interchangeably herein with
the term "fluorescent
reporter".
As used herein "quencher" or "quencher molecule" refers to a molecule that,
when
linked to a fluorescent probe comprising a fluorophore, is capable of
accepting the energy emitted by the
fluorophore, thereby quenching the emission of the fluorophore. A quencher can
be fluorescent, which
releases the accepted energy as light, or non-fluorescent, which releases the
accepted energy as heat, and
can be attached at any location along the length of the probe.
As used herein "dark quencher" refers to a non-fluorescent quencher.
As used herein, "probe" refers to an oligonucleotide that is capable of
forming a duplex
structure with a sequence in a target nucleic acid, due to complementarity of
at least one sequence of the
probe with a sequence in the target region, or region to be detected. The term
"probe" includes an
oligonucleotide as described above, with or without a fluorophore and a
quencher molecule attached.
The term "fluorescent probe" refers to a probe comprising a fluorophore and a
quencher molecule.
As used herein, "FAM" refers to the fluorophore 6-carboxy-fluorescein.
As used herein "JOE" refers to the fluorophore 6-carboxy-4',5'-dichloro-2',7'-
dimethoxyfluorescein.
As used herein, "TET" refers to the fluorophore 5-tetrachloro-fluorescein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the sequence of the oligonucleotide primers used in the real-
time
multiplex PCR reactions.
FIGURE 2 shows the sequence of the oligonucleotide probes used in the real-
time
multiplex PCR reactions. Each probe was covalently linked on its 5' end to the
FAMTM or TETTM
fluorophore.
FIGURE 3 shows the sensitivity of a HPV33 duplex PCR assay, described herein.
Results (mean SD, n=3) obtained with each specific probe are depicted by
different symbols: dark
circles represent a HPV33E6-FAM probe and white circles represent a HPV33E7-
TET probe.
FIGURE 4 shows the sensitivity of a HPV35 duplex PCR assay. Results (mean
SD,
n=3) obtained with each specific probe are depicted by different symbols: dark
circles represent a HPV35
E6-FAM probe and white circles represent a HPV35E7-TET probe.
FIGURE 5 shows the sensitivity of a HPV39 duplex PCR assay. Results (mean
SD,
n=3) obtained with each specific probe are depicted by different symbols: dark
circles represent a HPV39
E6-FAM probe and white circles represent a HPV35E9-TET probe.
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FIGURE 6 shows the sensitivity of a HPV51 duplex PCR assay. Results (mean
SD,
n=3) obtained with each specific probe are depicted by different symbols: dark
circles represent a HPV51
E6-FAM probe and white circles represent a HPV51E9-TET probe.
FIGITRE 7 shows the sensitivity of a HPV56 duplex PCR assay. Results (mean +
SD,
n=3) obtained with each specific probe are depicted by different symbols: dark
circles represent a HPV56
E6-FAM probe and white circles represent a HPV56E9-TET probe.
FIGURE 8 shows the sensitivity of a HPV59 duplex PCR assay. Results (mean +
SD,
n=3) obtained with each specific probe are depicted by different symbols: dark
circles represent a HPV59
E6-FAM probe and white circles represent a HPV59E9-TET probe.
FIGURE 9 shows the sensitivity of a HPV35 duplex PCR assay using a serial
dilution of
viral DNA purified from a human clinical specimen. Results (mean + SD, n=3 )
obtained with each
specific probe are depicted by different symbols: dark circles represent a
HPV35 E6-FAM probe and
white circles represent a HPV35E7-TET probe.
FIGURE 10 shows the sensitivity of a HPV39 duplex PCR assay using a serial
dilution
of viral DNA purified from a human clinical specimen. Results (mean SD, n=3)
obtained with each
specific probe are depicted by different symbols: dark circles represent a
HPV39 E6-FAM probe and
white circles represent a HPV39E7-TET probe.
FIGURE 11 shows the sensitivity of a HPV51 duplex PCR assay using a serial
dilution
of viral DNA purified from a human clinical specimen. Results (mean SD, n=3)
obtained with each
specific probe are depicted by different symbols: dark circles represent a
HPV51 E6-FAM probe and
white circles represent a HI'V51E7-TET probe.
FIGURE 12 shows the sensitivity of a HPV56 duplex PCR assay using a serial
dilution
of viral DNA purified from a human clinical specimen. Results (mean SD, n=3)
obtained with each
specific probe are depicted by different symbols: dark circles represent a
HI'V56 E6-FAM probe and
white circles represent a HPV56E7-TET probe.
FIGURE 13 shows the sensitivity of a HPV59 duplex PCR assay using a serial
dilution
of viral DNA purified from a human clinical specimen. Results (mean SD, n=3
) obtained with each
specific probe are depicted by different symbols: dark circles represent a
HPV59 E6-FAM probe and
white circles represent a HPV59E7-TET probe.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to an assay for individual detection of HPV types
HPV33, HPV35,
HPV39, HPV51, HPV56, and HI'V59 in a clinical sample, said types having been
associated with an
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oncogenic phenotype. Use of the assays of the present invention substantially
reduces the risk of false
negative results as compared to other assays known in the art.
It is well known that the relationship between the HPV genome and chromosomal
host
DNA may change during the multistage tumorigenic process (For review, see
McMurray et al., Int. J.
Exp. Path. 82: 15-33 (2001)). Premalignant lesions are often associated with
episomal forms of HPV
DNA while later-stage tumors typically have integrated HPV sequences. As a
result of the integration
correlated with advanced stages of disease progression, the open reading frame
of specific HPV genes,
such as the Ll locus,. may become disrupted. Such disruption of HPV gene
sequence may lead to false
negative results in assays designed to specifically detect the disrupted
sequence.
Therefore, a preferred embodiment of the present invention provides a method
for
identifying the presence of a specific HPV type in a sample, wherein said
method comprises
simultaneously detecting and amplifying a plurality of HPV genes of a single
HPV type. A sample is
considered positive for the HPV type if a majority of the plurality of the
HI'V genes are detected by the
methods of the present invention. Another preferred embodiment of the present
invention provides an
assay for the presence of a specific HPV type, wherein said assay comprises
simultaneously detecting
and amplifying two HPV genes of a single HPV type. A sample is considered
positive for the HPV type
if both of the genes are detected and HPV negative if none of the genes are
detected by the methods of
the present invention. Said assay reduces the risk of obtaining false negative
results associated with
assays that test for a single HPV locus. The method of the present invention
is highly specific and
reproducible.
The method of the present invention for detecting HPV types in a clinical
sample also
substantially reduces the risk of false positive results as compared to other
assays known in the art. Such
false positive results are caused by the high degree of homology among
specific HPV genes as compared
to the same HPV genes from a different HPV type. This level of homology makes
it difficult to design a
PCR assay that is specific for a single HPV type. When utilizing other methods
known in the art that
detect single loci, therefore, it is necessary to confirm positive results by
serially testing for the presence
of several loci of a single HPV-type. The further experimentation required to
verify positive results is
cumbersome and time-consuming. Establishment of the HPV status of a clinical
sample for four
different HPV types typically consumes 26-30 man-hours.
Unlike the methods available in the art, the present invention provides a
method for
simultaneously detecting and amplifying a plurality of distinct HPV genes of a
single HPV type selected
from the group consisting of: HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59;
thus substantially
reducing the occurrence of false positive results commonly associated with
single-locus assays.
Additionally, the assay of the present invention does not require serial
experimentation to confirm
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positive results and greatly reduces the man-hours required to determine the
BPV status of a sample. The
methods of the present invention are, therefore, adaptable to high throughput
screening of clinical
samples for the nucleic acid of specific HPV types. Said methods allow
screening for numerous samples
simultaneously, e.g. through use of a 96-well PCR foimat, but retain high
specificity and accuracy.
Another HPV real-time PCR assay has been described in the art that utilizes a
multiple
fluorophore format (Josefsson et al., Journal of Clinical Microbiology 37(3):
490-96 (1999)). This
method utilizes a mixture of specific and degenerate primers to amplify a
portion of the El gene in a
number of HPV types. Up to three probes were used per assay, each probe
comprising a different
fluorophore and each probe detecting the El gene of a different HPV type.
Assay sensitivity was tested
using plasmids containing HPV DNA and not in clinical samples.
Josefsson et al. disclose a substantially reduced sensitivity in detection of
HPV 18 DNA
when multiple fluorescent probes, each specific to a different HPV type, were
used simultaneously as
compared to a single-probe assay. Similarly, detection of HPV35 was somewhat
reduced when a mixture
of probes for HPV 16, HPV33 and HPV3 5 were used, as compared to a single
probe for HPV3 5.
Additionally, somewhat reduced sensitivity was observed at high copy numbers
when using a multiple
probe assay to detect HPV 16 and HPV31.
The method of the present invention utilizes a plurality of fluorescent
probes, each probe
comprising a fluorophore that emits energy at a unique emission maxima
relative to each other
fluorophore used in the particular assay. The assays provided herein are
highly specific and are capable
of detecting fewer than ten copies of HPV genomic DNA at two loci.
The linearity and sensitivity of each PCR assay of the present invention was
confirmed
using loci-specific plasmids at concentrations ranging from 10 to 106
copies/reaction (see FIGURES 3-
8). The HPV33, HPV35, HPV39, HPV51, BPV56, and HPV59 duplex PCR assays were
linear within
the range of 10 to 106 copies. The sensitivity of the HPV duplex PCR assays
for HPV35 (FIGURE 9),
HPV39 (FIGURE 10), HPV51 (FIGURE 11), HPV56 (FIGURE 12) and HPV59 (FIGURE 13)
was also
confirmed using viral DNA isolated from human clinical samples.
Tremendous assay sensitivity, as exhibited by the methods of the present
invention, is
critical in screening clinical samples where the copy number of HPV may be
low. Because the physical
manifestations of HPV infection are often covert and the latency period
prolonged, infection with HPV
may not be detected until the patient has been diagnosed with cervical
intraepithelial neoplasia (CIN),
which, if allowed to go untreated, can progress to carcinoma. Typically,
higher grade lesions (CIN2,
CIN3 and carcinoma) are associated with high HPV copy number, which may be
detectable by traditional
methods known in the art. However, many assays currently in use are not
sensitive or specific enough to
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detect low copy number HPV. Tremendous sensitivity is critical, therefore, for
early detection of HPV
when HPV copy numbers are low and therapeutic intervention is more likely to
be effective.
The present invention more specifically relates to a method for detecting the
presence of
a human papillomavirus (HI'V) type in a nucleic acid-containing sample
comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and a
plurality
of oligonucleotide sets to produce a plurality of PCR amplicons;
wherein each oligonucleotide set consists of (a) a forward discriminatory PCR
primer
hybridizing to a first location of a nucleic acid sequence of an HPV type, (b)
a reverse discriminatory
PCR primer hybridizing to a second location of the nucleic acid sequence of
the HPV type downstream
of the first location, and (c) a fluorescent probe labeled with a quencher
molecule and a fluorophore
which emits energy at a unique emission maxima; said probe hybridizing to a
location of the nucleic acid
sequence of the HPV type between the first and the second locations;
wherein each oligonucleotide set specifically hybridizes to a different HPV
amplicon
derived from the same HPV type, and wherein the HPV type is selected from the
group consisting of
HPV33, HI'V35, HPV39, HPV51, HPV56, and HPV59;
allowing said nucleic acid polymerase to digest each fluorescent probe during
amplification to dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HI'V type if a change of
fluorescence is
detected in at least two emission maxima.
In a preferred embodiment of this invention, each oligonucleotide set of the
plurality of
oligonucleotide sets is specific to a single gene of the BPV type to be
detected. In other words, each
oligonucleotide set of the method of the present invention hybridizes to
nucleotide sequences derived
from a single HPV gene of the same type. For example, the oligonucleotide
primers and probe of a first
oligonucleotide set hybridize to the E6 gene,'the oligonucleotide primers and
probe of a second
oligonucleotide set hybridize to the E7 gene and the oligonucleotide primers
and probe of a third
oligonucleotide set hybridize to the L1 gene. As a result, a plurality of PCR
amplicons is created
wherein each PCR amplicon is specific to a single HPV gene of the HPV type to
be detected.
In an alternative embodiment of this invention, the forward discriminatory PCR
primer
and the reverse discriminatory PCR primer of at least one oligonucleotide set
are specific to a different
gene of the same HPV type. For example, a forward discriminatory primer
hybridizes to the E6 gene and
a reverse discriminatory priiner hybridizes to the E7 gene. As a result, at
least one PCR amplicon
comprises a sequence of nucleotides derived from more than one gene. The
oligonucleotide probe
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specific to said amplicon may hybridize, for example, to a sequence of
nucleotides derived from the E6
gene, a sequence of nucleotides derived from the E7 gene, or a sequence of
nucleotides that crosses the
E6/E7 boundary.
The change in fluorescence can be detected by an automated fluorometer
designed to
perform real-time PCR having the following features: a method of excitation to
excite the fluorophore of
the fluorescent probe, a means for heating and cooling PCR reaction mixtures
and a means for detecting
a change in fluorescence. This combination of features, when performed by a
single real-time PCR
instrument, allows real-time detection of PCR amplicons, which allows
confirmation of PCR product
amplification through examination of the kinetics of the fluorescence increase
in real-time. Automated
fluorometers for performing real time PCR reactions are known in the art and
can be adapted for use in
this specific assay, for example, the iCycler from Bio-Rad Laboratories
(Hercules, CA), the
Mx3000PTM, the MX3005PTM and the MX4000 from Stratagene (La Jolla, CA), the
ABI PRISM
7300, 7500, 7700, and 7900 Sequence Detection Instruments (Applied Biosystems,
Foster City, CA), the
SmartCycler and the Gene Xpert System (Cepheid, Sunnyvale, CA) and the
LightCycler (Roche
Diagnostics Corp., Indianapolis, IN).
The methods of the present invention were performed with an ABI PRISM 7700
Sequence Detection Instrument (Applied Biosystems). This instrument uses a
spectrograph to separate
the fluorescent emission (based on wavelength) into a predictably spaced
pattern across a charged-
coupled device (CCD) camera. A Sequence Detection System application of the
ABI PRISM 7700
collects the fluorescent signals from the CCD camera and applies data analysis
algorithms.
Nucleic acid polymerases for use in the methods of the present invention must
possess 5'
- 3' exonuclease activity. Several suitable polymerases are known in the art,
for example, Taq (Thei=nzus
aquaticus), Tbr (Therjnus brockianus) and Tth (Therinus tlzernaaphilus)
polymerases. TAQ DNA
polymerase is the preferred polymerase of the present invention. The 5' - 3'
exonuclease activity is
characterized by the degradation of double-stranded DNA encountered during
extension of the PCR
primer. A fluorescent probe annealed to the amplicon will be degraded in a
similar manner, thus
releasing the fluorophore from the oligonucleotide. Upon dissociation of the
fluorophore and the
quencher, the fluorescence emitted by the fluorophore is no longer quenched,
which results in a
detectable change in fluorescence. During exponential growth of the PCR
product, the amplicon-specific
fluorescence increases to a point at which the sequence detection application,
after applying a
multicomponenting algorithm to the composite spectrum, can distinguish it from
the background
fluorescence of non-amplifying samples. The ABI PRISM 7700 Sequence Detection
Instrument also
comprises a software application, which determines the threshold cycle (Ct)
for the samples (cycle at
which this fluorescence increases above a pre-determined threshold). PCR
negative samples have a Ct
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equal to the total number of cycles performed and PCR positive samples have a
Ct less than the total
number of cycles performed.
The present invention relates to a method for detecting the presence of a
human
papillomavirus (HPV) type in a nucleic acid-containing sample, wherein the HPV
type is selected from
the group consisting of: HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59. In a
preferred
embodiment of the method of the present invention, the number of
oligonucleotide sets is two and the
sample is positive for the HPV type tested if a change of fluorescence is
detected in both fluorophores.
In a further preferred embodiment of the method of the present invention, the
oligonucleotide sets specifically hybridize to the E6 and E7 genes of HPV. A
sample is positive for the
HPV type being tested if both the E6 and E7 genes are detected.
Oligonucleotide probes and primers of the present invention can be synthesized
by a
number of methods. See, e.g., Ozaki et al., Nucleic Acids Research 20: 5205-
5214 (1992); Agrawal et
al., Nucleic Acids Research 18: 5419-5423 (1990). For example, oligonucleotide
probes can be
synthesized on an automated DNA synthesizer such as the ABI 3900 DNA
Synthesizer (Applied
Biosystems, Foster City, CA). Alternative chemistries, e.g. resulting in non-
natural backbone groups,
such as phosphorothioate, phosphoramidate, and the like, may also be employed
provided that the
hybridization efficiencies of the resulting oligonucleotides are not adversely
affected.
The PCR amplification step of the present invention can be performed by
standard
techniques well known in the art (See, e.g., Sambrook, E.F. Fritsch, and T.
Maniatis, Molecular Cloniizg:
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press
(1989); U.S. Patent No.
4,683,202; and PCR Protocols: A Guide to Methods andApplications, Innis et
al., eds., Academic Press,
Inc., San Diego (1990) which are hereby incorporated by reference). PCR
cycling conditions typically
consist of an initial denaturation step, which can be performed by heating the
PCR reaction mixture to a
temperature ranging from about 80 C to about 105 C for times ranging from
about 1.to about 15 min.
Heat denaturation is typically followed by a number of cycles, ranging from
about 20 to about 50 cycles,
each cycle usually comprising an initial denaturation step, followed by a
primer annealing/primer
extension step. Enzymatic extension of the primers by the nucleic acid
polymerase, e.g. TAQ
polymerase, produces copies of the template that can be used as templates in
subsequent cycles.
"Hot start" PCR reactions may be used in conjunction with the methods of the
present
invention to eliminate false priming and the generation of non-specific
amplicons. To this end, in a
preferred embodiment of this invention, the nucleic acid polymerase is
AmpliTaq Gold (Roche
Molecular Systems, Pleasanton, CA) DNA polymerase and the PCR cycling
conditions include a "hot
start" PCR reaction. Said polymerase is inactive until activation, which can
be accomplished by
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incubating the PCR reaction components at 95 C for approximately 15 minutes
prior to PCR cycling.
PCR methods comprising a similar initial incubation step are known in the art
as "hot start" PCR assays.
Preferably, oligonucleotide probes of the present invention are in the range
of about 20
to about 40 nucleotides in length. More preferably, the oligonucleotide probe
is in the range of about 18
to about 30 nucleotides in length. Most preferably, the oligonucleotide probe
is in the range of about 24
to about 30 nucleotides in length. The precise sequence and length of an
oligonucleotide probe of the
invention depends in part on the nature of the target polynucleotide to which
it binds. The binding
location and length may be varied to achieve appropriate annealing and melting
properties for a particular
embodiment.
Preferably, the 3' tenninal nucleotide of the oligonucleotide probe is blocked
or rendered
incapable of extension by a nucleic acid polymerase. Such blocking is
conveniently carried out by
phosphorylation of the 3' terminal nucleotide, since the DNA polymerase can
only add nucleotides to a
3' hydroxyl and not a 3' phosphate.
It is preferred that HPV primers and probes of the present invention do not
share full
homology with other HPV types. Each primer of the present invention should be
designed so that 3'
homology is lacking in at least one nucleotide or more. Such primer design
would substantially reduce
the chance of the primer annealing to the wrong HPV type and prevent primer
extension if annealing to
an HPV type that was not intended does occur since TAQ DNA Polymerase only
extends a primer from
the 3' end and requires that the 3' end be properly annealed.
It is also preferred that each probe contain mismatches along the length of
the
oligonucleotide which destabilize the oligonucleotide binding to non-specific
HPV targets. As few as
one mismatch along the length of the oligonucleotide probe is enough to
discriminate between loci.
Because the probe of the present invention is only hydrolized and detected
when bound to the segment of
DNA that is being amplified, non-specific binding of the probe to a DNA
sequenced that is not being
amplified is not detected.
To this end, the present invention relates to a primer pair for the PCR
amplification of
HPV nucleic acid, wherein both the forward and reverse PCR primers are
discriminatory. In a preferred
embodiment of the invention, the nucleotide sequences of the primer pair are
selected from the group
consisting of: SEQ ID NO:1 and SEQ II) NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ
ID NO:5 and
SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ II) NO:9 and SEQ ID NO:10, SEQ
ID NO:11 and
SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16,
SEQ ID NO:17
and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID
NO:22, and SEQ
ID NO:23 and SEQ ID NO:24.
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It is readily apparent to those skilled in the art that other discriminatory
oligonucleotide
primers may be designed that selectively amplify HPV genes of a specific type.
Said oligonucleotide
primers may be the same length as those disclosed herein or may be in the
range of 12-45 nucleotides.
More preferably, the length of the oligonucleotide primers of the present
invention is in the range of 18-
30 nucleotides. Most preferably, the lengtli of the oligonucleotide primers of
the present invention is in
the range of 19-29 nucleotides.
It is also preferred that each probe contain mismatches along the length of
the
oligonucleotide which destabilize the oligonucleotide binding to non-specific
HPV targets. As few as
one mismatch along the length of the oligonucleotide probe is enough to
discriminate between loci.
Because the probes of the present invention are only hydrolized and detected
when bound to the segment
of DNA that is being amplified, non-specific binding of the probe to a DNA
sequenced that is not being
amplified is not detected.
To this end, a preferred embodiment of this invention relates to an
oligonucleotide probe
comprising a sequence of nucleotides specific to a single HPV type. Said
oligonucleotide probe can bind
to specific HPV amplicons resulting from PCR amplification of viral DNA using
specific oligonucleotide
primers. In a further embodiment of this invention, said oligonucleotide probe
comprises a sequence of
nucleotides selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 1, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID
NO:34, SEQ ID NO: 35, and SEQ ID NO:36. The present invention also relates to
said oligonucleotide
probes further comprising a fluorophore and a quencher molecule.
The fluorophores of the present invention may be attached to the probe at any
location of
the probe, including the 5' end, the 3' end or internal to either end, i.e.
said fluorophore may be attached
to any one of the nucleotides comprising the specific sequence of nucleotides
capable of hybridizing to
the specific I-IPV gene that the probe was designed to detect. In a preferred
embodiment of this
invention, the fluorophore is attached to a 5' terminal nucleotide of the
specific sequence of nucleotides
and the quencher is attached to a 3' terminal nucleotide of the specific
sequence of nucleotides.
Preferably, fluorophores are fluorescent organic dyes derivatized for
attachment to the 3'
carbon or terminal 5' carbon of the probe via a linking moiety. Preferably,
quencher molecules are also
organic dyes, which may or may not be fluorescent, depending on the embodiment
of the invention. For
example, in a preferred embodiment of the invention, the quencher molecule is
non-fluorescent.
Generally, whether the quencher molecule is fluorescent or simply releases the
transferred energy from
the reporter by non-radiative decay, the absorption band of the quencher
should substantially overlap the
fluorescent emission band of the reporter molecule. Non-fluorescent quencher
molecules that absorb
energy from excited reporter molecules; but which do not release the energy
radiatively, are referred to
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herein as "dark quenchers," "dark quencher molecules," "non-fluorescent
quenchers" or "non-fluorescent
quencher molecules".
Several fluorophore-quencher pairs are described in the art. See, e.g. Pesce
et al, editors,
Fluorescence Spectroscopy, Marcel Dekker, New York, (1971); White et al,
Fluorescence Analysis: A
Practical Approach, Marcel Dekker, New York, (1970); and the like. The
literature also includes
references providing exhaustive lists of fluorescent and non-fluorescent
molecules and their relevant
optical properties, e.g. Berlman, Handbook of Fluorescence Sprectra
ofAroinatic Molecules, 2nd
Edition, Academic Press, New York, (1971). Further, there is extensive
guidance in the literature for
derivatizing reporter and quencher molecules for covalent attachment via
common reactive groups that
can be added to an oligonucleotide. See, e.g. U.S. Pat. No. 3,996, 345; and
U.S. Pat. No. 4,351,760.
Exemplary fluorophore-quencher pairs may be selected from xanthene dyes,
including
fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are
widely available
commercially with substituents on their phenyl moieties which can be used as
the site for bonding or as
the bonding functionality for attachment to an oligonucleotide. Another group
of fluorescent compounds
are the naphthylamines, having an amino group in the alpha or beta position.
Included among such
naplithylamino compounds are 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-
naphthalene sulfonate
and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7-
isocyanatocoumarin, acridines,
such as 9- isothiocyanatoacridine and acridine orange; N-(p-(2-
benzoxazolyl)phenyl)maleimide;
benzoxadiazoles, stilbenes, pyrenes, and the like.
Preferably, fluorophore and quencher molecules are selected from fluorescein
and
rhodamine dyes. These dyes and appropriate linking methodologies for
attachment to oligonucleotides
are known in the art. See, e.g. Marshall, Histochemical J. 7:299-3 03 (1975);
and U.S. Pat. No.
5,188,934. In a preferred embodiment of this invention, the fluorophores are
selected from the group
consisting of: 6-carboxy-fluorescein (FAMTM, Applera Corp., Norwalk, CT), 6-
carboxy-4',5'-dichloro-
2',7'-dimethoxyfluoreseein (JOETM, Applera Corp.), 5-tetrachloro-fluorescein
(TETTM, Applera Corp.),
and CAL fluorD Orange (BioSearch Technologies Inc., Novato, CA).
Other fluorophores for use in the methods of the present invention include,
but are not
limited to: CAL fluor red (BioSearch Technologies Inc.), VICTM and HEXTM
(Applera Corp., Norwalk,
CT), Texas Red (Molecular Probes, Inc., Eugene, Oregon), Yakima Yellow
(Epoch Biosciences, Inc.,
Bothell, WA), and Cy3 and Cy5 (Amersham Biosciences, Piscataway. NJ).
In a preferred embodiment of this invention, the quencher molecule is non-
fluorescent
(dark quencher). Dark quenchers bave a lower background fluorescence and do
not emit light, allowing
additional fluorophore options for multiplex assays. A preferred quencher
molecule of the present
invention is Black Hole Quencher~M 1(BHQl), a non-fluorescent quencher
developed by Biosearch
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Technologies (Novato, CA). Other dark quenchers include, but are not limited
to: BHQTM-2, BHQTM-3
(Biosearch Tech.), Eclipse Dark Quencher (Epoch Biosciences, Inc., Bothell,
WA), and Deep Dark
QuencherTM I and II ((DDQ) Eurogentec s.a., Seraing, Belgium). Although dark
quenchers are preferred
for use in the present invention, one of skill in the art could select a
fluorescent quencher for use in the
methods of the present invention; for example, 6-carboxy-tetramethyl-rhodamine
(TAMRATM, Applera
Corp., Norwalk, CT), providing that said fluorescent quencher does not
interfere with detection of the
energy emitted by each of the chosen fluorophores.
Optimal quenchers for use in the methods of the present invention are selected
based on
their ability to quench the fluorescence of a selected fluorescent dye, said
dye emitting energy in the form
of light with a defmed spectrum. One of skill in the art can readily identify
a fluorophore-quencher pair
for use in the methods of the present invention. Preferred fluorophore-
quencher pairs include: FAM-
BHQ1, JOE-BHQ1, and TET-BHQ1. Additional fluorophore-quencher pairs described
in the art include:
Cy3-BHQ2, Cy5-BHQ3, TET-TAMRA, HEX-TAMRA, Texas Red-DDQ I or 11. One of skill
in the art
will realize that the particular quencher chosen must be capable of
effectively quenching the fluorescence
of the chosen fluorophore at the wavelength said fluorescence is emitted. One
of skill in the art will also
realize that when choosing multiple fluorophores suitable for the purpose of
simultaneous detection of
various templates (multiplexing), each fluorophore should emit energy at a
unique emission maxima.
Preferably, commercially available linking moieties are employed that can be
attached to
an oligonucleotide during synthesis, e.g. available from Clontech Laboratories
(Palo Alto, Calif.).
The present invention relates to a method for detecting the presence of BPV33
nucleic
acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 1, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:2, and a probe as
set forth in SEQ ID NO:25, said probe labeled with a quencher moleule on the
3' end and a fluorophore
on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:3, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:4, and a probe as
set forth in SEQ ID NO:26, said probe labeled with a quencher molecule on the
3' end and a fluorophore
on the 5' end;
allowing said nucleic acid polymerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
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detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
detennining that the sample is positive for the HPV33 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: FAM, JOE and TET, and the quencher molecule is
BHQ1.
In a further preferred embodiment of the method for detecting the presence of
HPV33 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
The present invention further relates to a method for detecting the presence
of HPV35
nucleic acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:5, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:6, and a probe as
set forth in SEQ ID NO:27, said probe labeled with a quencher molecule on the
3' end and a fluorophore
on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:7, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:8, and a probe as
set forth in SEQ ID NO:28, said probe labeled with a quencher molecule on the
3' end and a fluorophore
on the 5' end;
allowing said nucleic acid polyrnerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV35 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: F.AM, JOE and TET, and the quencher is BHQ1.
In a further preferred embodiment of the method for detecting the presence of
HPV35 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
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The present invention is also related to a method for detecting the presence
of HPV39
nucleic acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:9, a reverse discriminatory PCR primer as set forth in SEQ
ID NO: 10, and a probe
as set forth in SEQ ID NO:29, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 11, a reverse discriminatory PCR primer as set forth in
SEQ ID NO: 12, and a probe
as set forth in SEQ ID NO:30, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
allowing said nucleic acid polymerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV39 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: FAM, JOE and TET, and the quencher is BHQl.
In a fitrther preferred embodiment of the method for detecting the presence of
HPV39 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
This invention additionally relates to a method for detecting the presence of
HPV51
nucleic acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 13, a reverse discriminatory PCR primer as set forth in
SEQ ID NO: 14, and a probe
as set forth in SEQ ID NO:3 1, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 15, a reverse discriminatory PCR primer as set forth in
SEQ ID NO: 16, and a probe
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as set forth in SEQ ]D NO:32, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
allowing said nucleic acid polymerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV51 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: FAM, JOE and TET, and the quencher is BHQ1.
In a further preferred embodiment of the method for detecting the presence of
HPV51 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
This invention additionally relates to a method for detecting the presence of
HPV56
nucleic acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 17, a reverse discriminatory PCR primer as set forth in
SEQ ID NO: 1S, and a probe
as set forth in SEQ ID NO:33, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO: 19, a reverse discriminatory PCR primer as set forth in
SEQ ID NO:20, and a probe
as set forth in SEQ ID NO:34, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
allowing said nucleic acid polymerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV56 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: FAM, JOE and TET, and the quenclier is BHQ1.
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In a further preferred embodiment of the method for detecting the presence of
HPV56 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
This invention further relates to a method for detecting the presence of HPV59
nucleic
acid in a nucleic acid-containing sample comprising:
amplifying the nucleic acid in the presence of a nucleic acid polymerase and
two
oligonucleotide sets;
the first oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:21, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:22, and a probe
as set forth in SEQ ID NO:35, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
the second oligonucleotide set consisting of a forward discriminatory PCR
primer as set
forth in SEQ ID NO:23, a reverse discriminatory PCR primer as set forth in SEQ
ID NO:24, and a probe
as set forth in SEQ ID NO:36, said probe labeled with a quencher molecule on
the 3' end and a
fluorophore on the 5' end;
allowing said nucleic acid polymerase to digest each probe during
amplification to
dissociate said fluorophore from said quencher molecule;
detecting a change of fluorescence upon dissociation of the fluorophore and
the
quencher, the change of fluorescence corresponding to the occurrence of
nucleic acid amplification; and
determining that the sample is positive for the HPV59 type if a change of
fluorescence is
detected with the two probes.
In a preferred embodiment of the method described above, the fluorophore is
selected
from the group consisting of: FAM, JOE and TET, and the quencher is BHQ 1.
In a further preferred embodiment of the method for detecting the presence of
HPV59 in
a sample described above, the fluorophore of the first oligonucleotide set is
FAM and the fluorophore of
the second oligonucleotide set is TET.
Having described preferred embodiments of the invention with reference to the
accompanying drawings, it is to be understood that the invention is not
limited to those precise
embodiments, and that various changes and modifications may be effected
therein by one skilled in the
art witllout departing from the scope or spirit of the invention as defined in
the appended claims.
The following examples illustrate, but do not limit the invention.
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EXAMPLE 1
Discriminatory HPV Primer Design
PCR primers were designed for each HPV type using Primer Express v. 1.0 (PE
Applied
Biosystems, Foster City, CA). The gene-specific nucleotide sequences of the
open-reading frames of the
E6 and E71oci of the HPV33, HPV35, FIPV39, HPV51, HPV56 and HPV59, types were
aligned using
ClustalW v.1.7 (European Molecular Biology Laboratory, Heidelberg, Germany)
and a Power Macintosh
G4 personal computer (Apple Computer). The Phylip-format alignment file was
then imported into the
Allelic Discrimination module of the Primer Express application and the
specific HPV type was marked.
Primer pairs were selected that met the following criteria: Tm = 59-61 C,
amplicon size:
100-250 bp, GC content between 20-80%, a guanosine or cytosine residue at the
3'-terminal position, and
the discriminatory base within the three 3'-terminal bases. The discriminatory
base is the residue that is
unique for the specific HPV type at the specific position and acts to
discriminate the HPV type from the
others in the alignment. Several primer pairs were selected such that both the
sense and antisense
primers were discriminatory (see FIGURE 1).
The primer sequences were analyzed for uniqueness and primer-diiner formation
by
Aniplify v. 1.2 for Macintosh (William Engels, Genetics Department, University
of Wisconsin). An
optimal primer pair was selected for each loci in which there was no apparent
dimer formation and, each
primer was predicted to anneal to one and only one location of the target
loci. Once an amplicon was
defined by a primer pair, a dual-labeled oligonucleotide probe was designed
that met the following
criteria: Tm = 68-70 C, length <30 nt, runs of no more than three of the same
nucleotide, no guanosine
residue on the 5' terminus and more cytosine residues than guanosine residues
(see FIGURE 2).
The predicted cross-reactivity of each primer and probe to other known BPV
types was
assessed by BLAST searching each sequence against the NCBI Genbank database.
Most primer and
probe sequences returned unique hits for the specific HPV for which they were
designed and did not
share any homology with other HPV types. The HPV33E7 antisense primer shares
some homology with
HPV52, HPV67, and HPV58. The HPV35E6 antisense primer shares some homology
with BPV16. The
HPV35 probe shares some homology with HPV16. The HPV35E7 sense primer shares
some homology
with HPV16, HPV31, HPV33, HPV58, and HPV67. The HPV35E7 sense primer shares
some homology
with HPV31 and HPV67. The HPV39E7 TaqMan probe shares some homology with
HPV70. The
HPV39 E7 antisense primer shares some homology with HPV59. The HPV51E6 sense
primer shares
some homology with HPV82. The HPV51 E6 TaqMan probe shares some hoinology with
HPV82. The
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HPV51 E7 sense primer shares some homology with HPV26 and HPV82. The HPV51 E7
Taqman probe
shares some homology with HPV26
None of the HPV primers and probes that were designed share full homology with
other
HPV types. Each primer lacks 3' homology of at least one nucleotide or more
which suggests that even
if it were to anneal to the wrong HPV type, it would not be extended since TAQ
DNA Polymerase only
extends a primer from the 3' end and requires that the 3' end be properly
annealed. Each TaqMan probe
contains mismatches along the length of the oligonucleotide which destabilize
the oligonucleotide
binding to non-specific targets. As few as one mismatch along the length of
the oligonucleotide probe is
enough to discriminate between loci. In addition, the probe is only hydrolized
and detected when bound
to the segment of DNA that is being amplified. Non-specific binding of the
probe to a DNA sequenced
that is not being amplified is not detected.
EXAMPLE 2
Synthesis and Labeling of Oligonucleotide Primers and Probes
The oligonucleotide primers were custom synthesized and reverse-phase BPLC-
purified
by Operon Technologies (Huntsville, AL). The dual-labeled oligonucleotide
probes were custom
synthesized and reverse-phase HPLC-purified by Biosearch Technologies (Novato,
CA). The
oligonucleotide fluorescent probes for the E6 loci were 5'-labeled with 6-
carboxy-fluorescein (FAM), the
oligonucleotide fluorescent probes for the E71oci were 5'-lableled with 5-
tetrachloro-fluorescein (TET),
available from Molecular Probes (Eugene, OR). All oligonucleotide probes were
3'-labeled with
BHQTMI, a non-fluorescent quencher developed by Biosearch Technologies
(Novato, CA). The
lyophilized primers and probes were reconstituted in 1X TE pH 8.0 buffer
(Roche Molecular
Biochemicals) and the concentration determined by measuring the O.D. at 260 nm
on a Beckman 600DU
spectrophotometer and calculating the concentration using the oligonucleotide-
specific molar extinction
coefficient.
EXAMPLE 3
Optimization of the Multiplex Reaction
Primer and probe concentrations were optimized so that three separate loci
could be
simultaneously detected and amplified in a single PCR tube without favoring
one reaction over another.
The fluorescent oligonucleotide probe concentrations were optimized separately
by assessing the
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threshold cycle (Ct) and ARn of increasing probe concentrations using 100
copies of DNA template (each
locus cloned into a plasmid) on the ABI PRISM 7700 Sequence Detection System
instrument.
Samples were amplified in a 50 L reaction mixture containing 25 L of the
TaqMan
Universal PCR 2X PCR Master Mix (Applied Biosystems, Foster City, CA), 200 nM
final concentration
of each primer, 100 copies of plasmid DNA template, DEPC-treated water
(Ambion) and a range of
concentrations (25-200 nM) of fluorescently-labeled oligonucleotide probes.
The cycling conditions
consisted of an initial step of 50 C for 2 min followed by 95 C for 10 min,
and 45 cycles of 94 C for 15
sec and 60 C for 1 min.
Included in the Taq-Man Universal PCR master mix is dUTP (instead of dTTP) and
uracil-N-glycosylase (UNG), an enzyme that is activated at 50 C and cleaves
uracil-containing nucleic
acids. See Longo et al., Gene 93: 125-128 (1990). UNG prevents the
reamplification of carryover PCR
products in subsequent experiments.
A concentration of each probe was selected that exhibited the lowest Ct and a
ARn - 1.
The primer concentrations were optimized for each locus by assessing the Ct
and ARn of each primer
concentration combination in a fine matrix assay using the previously
determin.ed concentration of loci-
specific oligonucleotide probe and ten copies of the plasmid DNA template. The
concentrations of the
sense and antisense primers that exhibited the lowest Ct and maximal ARn were
selected.
The primers and probes were then tested together with the addition of extra
AmpliTaq
Gold DNA Polymerase (0.75 U/well, Applied Biosystems, Foster City, CA). The
additional DNA
polymerase was added because the TaqMan Universal 2X PCR Master Mix, which
already contains
AmpliTaq Gold DNA Polymerase, was optimized for duplex reactions and not for
triplex reactions. The
additional DNA polymerase supplements the DNA polymerase in the 2X master mix
and reinforces the
reaction.
The linearity and sensitivity of each PCR assay was confirmed using loci-
specific
plasmids at concentrations ranging from 10 to 106 copies/reaction. The HPV33,
HPV35, HI'V39,
HPV51, HPV56, and HPV59 multiplex PCR assays were linear witliin the range of
10 to 106 copies.
EXAMPLE 4
DNA Isolation
DNA was isolated from human clinical specimens using the QlAamp 96-well DNA
Spin
Blood Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol
with the following
modifications: the quantity of Qiagen protease was increased to 0.5 mg/well
instead of the recommended
0.4 mg/well, the QlAamp filter plate was centrifuged dry atop a clean square-
well block in a Sigma
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Centrifuge (Qiagen Inc, Valencia, CA) for 10 min. at 6000 RPM and the DNA was
eluted with pre-
warmed (70 C) elution buffer.
EXAMPLE 5
Screeningof Human Clinical Samples
A master mix containing all of the components of the PCR reaction except the
template
DNA was prepared and loaded into 96-well optical reaction plates (46 1 well,
Applied Biosystems,
Foster City, CA) for each HPV type being tested. Four l of the purified DNA
was added to each well
containing the Multiplex PCR master mix and the wells were capped with optical
PCR caps (Applied
Biosystems, Foster City, CA). After centrifugation at 3000 RPM for 2 min in a
Sigma centrifuge, the 96-
well PCR plate was transferred to the ABI PRISM 7700 Sequence Detection
Systems Instrument
(Applied Biosystems, Foster City, CA).
PCR cycling and data collection were initiated and controlled by a pre-
designed template
that is specific for each HPV type. When the PCR cycling was complete, the
data was saved
electronically and the amplification plate discarded. The data was then
analyzed using the Sequence
Detection Systems application (Applied Biosystems, Foster City, CA). The
thresholds for each dye layer
were manually set; the FAM dye layer threshold was set to 0.05 and the TET dye
layer was set to 0.04.
The data were then exported electronically to a tab-delimited text file. The
text file and the file
containing the sample names was imported into the HPV type-specific Microsoft
EXCEL workbook.
The locked worksheets contained embedded formulas which calculated dye layer
PCR positivity based
on the threshold cycle of each sample. Data from all three dye layers were
then compiled by the
workbook, which calculates a consensus HPV PCR positivity of each sample based
on the rules set
above.
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